The intra-day and inter-day precisions were evaluated at two different concentration levels (0.05 and 5 μg L−1 of dye) on the same day and five distinct days, respectively. The analytical utility of the absorbents packed in pulses and mini-columns to extract and recover EY dye was attained by 98.94%. The column could efficiently remove different dyes from real industrial effluents, and hence the sol-gel/PUF is a good competitor for commercial applications. The findings of this research work have strong potential in the future to be used in selecting the most suitable lightweight growing medium for a green roof based on stakeholder requirements. Therefore, this study has provided a convenient pathway for the preparation of compressible and reusable sponge materials from renewable biomass for efficient removal of EY from the water environment.Textile dyes widely used in industrial products are known as a major threat to human health and water ecological security. On the other hand, sol gel represents a principal driver of the adoption of dispersive solid-phase microextractors (d-µ SPME) for pollutants residues in water. Thus, the current study reports a new and highly rapid and highly efficient hybrid sol-gel-based sponge polyurethane foam as a dispersive solid-phase microextractor (d-µ-SPME) platform packed mini-column for complete preconcentration and subsequent spectrophotometric detection of eosin Y textile dye in wastewater. The unique porous structure of the prepared sol-gel immobilized polyurethane foams (sol-gel/PUF) has suggested its use for the complete removal of eosin Y dye (EY) from water. In the mini-column, the number (N) of plates, the height equivalent to the theoretical plates (HETP), the critical capacity (CC), and the breakthrough capacities (BC) of the hybrid sol-gel-treated polyurethane foams towards EY dye were determined via the breakthrough capacity curve at various flow rates. Under the optimum condition using the matrix match strategy, the linear range of 0.01–5 µg L In today’s era, freshwater demand has increased significantly, as it is regarded as an essential component of the earth’s ecosystem and plays an important role in modern life ,2. FreshRecently, eosin yellow (EY) textile dye has received great awareness because of its high solubility in water and its distinctive fluorescence . Eosin y2/chitosan-montmorillonite  abbreviated as MAPTMS  and the modified organic zirconium complex, prepared from the chelation of Zr4+-n-propoxide . ZPO was 80:20, and the theoretical hydrolysis degree was 50% against the total content of reactive alkoxide groups. The synthesis required a three-step process, as shown in the 3 (0.1 M) solution with a 1:0.25 ratio. The hydrolysis is performed in a heterogeneous way within 5 min until methanol production becomes sufficient to enable the miscibility of all species in solution. In parallel, ZPO is chelated with MAAH to block two of its alkoxide groups and minimize precipitation when it is in contact with water. These two reactions were achieved simultaneously and allowed to stir for 45 min, and the partially hydrolyzed MAPTMS was added gradually by drop-wise addition to the Zr (VI)-complex. Nutral hydrolysis was then performed after 5 min with deionized water by drop-wise addition to the mixture. A diagram describing the Sol gel preparation is demonstrated in the Electronic Supplementary Information’s (The sol-gel matrix was prepared via hydrolysis and condensation reactions of the silicon precursor 3-methacryloxypropyltrimethoxy–silane (Hmation’s .v/v) with constant stirring for 30 min. After shaking, the sol-gel-impregnated PUF cubes were then separated out and dried between sheets of filter paper, as mentioned earlier [An accurate mass (1.0 g) of the PUF cubes was dried and shaken with Sol-gel dissolved in isopropanol  ethanol, 0.5 M NaOH, and deionized water, respectively, at a 3.0 mL flow rate.A cylindrical polyethylene tube with a length of 8.0 cm and a 51.0 mm internal diameter served as a mini-column. The tube was manually packed with the Sol-gel/PUF under suction by fixing the end of the tube to the suction port of the SPE manifold. After effective packing, the suction was continued for 30 min to reduce channeling inside the mini-column. After completion of the task, the mini-columns were washed with 3% (−1. The dye solutions (100.0 mL) were percolated individually through the columns at various flow rates between 2, 5, 7, 12, 17, and 22 mL min−1. The retained species were then recovered from the sol-gel-treated PUF packed column with acetone (10 mL) and/or sodium hydroxide (1.0 M), a proper eluating agent, at a flow rate of 1.0–2.0 mL min −1. The absorbances of the recovered solutions were then measured against the reagent blank. The percentage recovery of the dye adsorption or the extracted dye content was estimated by comparing the concentration of the analyte before and after adsorption.In the univariate fixed-bed experiments of mini-column, the parameters assessed were the impact of flow rate and initial dye concentration on adsorption. The EY dye concentrations were 1, 5, 10, and 20 mg mL−1) of EY dye at the optimum conditions were passed through the sol-gel/PUF-packed mini-column (0.5g ± 0.002) at 5, 10, and 20 mL min−1 flow rates. This was carried out until the concentration of the tested EY dye in the effluent reached that of the feed one. The concentration of the tested EY dye was determined from the absorbance measurement of the effluent at λmax versus reagent blank for each tested EY dye. The critical capacity (CC), the breakthrough capacity (BC), the number (N), and the height equivalent of the theoretical plates (HETP) were finally determined as reported earlier [In this experiment, various fractions (200 mL) of the test feed aqueous solution (3.5 L) containing , was percolated through sol-gel immobilized PUF-packed columns at 7.0 mL min−1 flow rate under the optimized parameters of dye retention. Complete retention of the EY dye was achieved, as detected by measuring the dye in the effluent solution. Attempts to recover the dye from the packed were performed using a variety of eluating agents, e.g., NaOH , acetone, and acetone-NaOH (1.0 mol L−1) at a 1:1 ratio (v/v) individually at a 0.25 mL min −1 flow rate. Various fractions (250 μL) of the eluating agent at a 0.25 mL min−1 flow rate were percolated through the sorbent-packed column. The absorbance of the eluated fractions (eluate) was determined at the optimized wavelength against a reagent blank.The analytical performance of the sol-gel/PUF-packed (0.3 g ± 0.002) mini-column was also calculated by the chromatogram (Gluenkauf method) as follows: An aqueous solution (100 mL), individually spiked with EY  packed mini-columns. Blank experiments were also performed in the absence of the tested compounds. The retained EY dye was recovered with NaOH  and acetone (5 mL). The solution was finally adjusted to pH ≤ 3.0 with NaOH, and the absorbance of the total effluent was measured at λmax against the reagent blank.Amounts of 500 mL of tap water, sea water, and industrial water were filtered through a 0.45 µm filter membrane and stored in low-density polyethylene (LDPE) bottles. The procedure was carried out on samples containing EY dye at concentrations in the ranges 0.05–5.0 mg ml


This study aimed to establish the survival rate of unilateral removable partial dentures (u-RPD) comparative with bilateral RPD (bi-RPD) with major connector in elder patients, as well as to determine both their treatment satisfaction and oral health.The study sample included 17 patients treated with u-RPD and 17 patients treated with bi-RPD with a major connector. The patients were followed over five years with recalls every 6 months. A 5- points Likert scale was used to determine the satisfaction of the patients. The Oral Health Impact Profile-14 (OHIP-14) questionnaire was used to evaluate their oral health after each type of administrated treatment. The local oral examined aspects included the maintenance of the abutment teeth periodontal health, the fractures of the removable dentures, the fractures of the connectors, the chipping of the aesthetic material. Kaplan–Meier survival analysis was conducted to evaluate the performance of the two treatments.The mean survival time in years was 4.882 ± 0.114, 95% CI  and 4.882 ± 0.078, 95% CI , for the u-RPD and the bi-RPD, respectively. The five-year survival rates for the two dentures were 94.1% for u-RPD vs. 88.2% for bi-RPD with a major connector, without a statistically significant difference between them (Log-rank test χ2(1) = 0.301, p = 0.584). The patients receiving u-RPD presented significantly higher satisfaction scores compared to the patients receiving bi-RPD, 4.88 ± 0.48 vs. 4.41 ± 0.62, Mann-Whitney U test, p = 0.026.Patients receiving u-RPD presented higher levels of treatment satisfaction and better oral health than patients receiving bi-RPD. The survival rates of the treatments u-RPD and bi-RPD were similar. Due to an increasing aging population retaining more teeth at an older age, in the era of fixed partial dentures, the removable partial dentures (RPD) are still used to restore edentate patients. Psychological factors are important in patients suffering from tooth loss and/or in those awaiting prosthetic care with fixed or removable dentures , 2. AlsoIt would be correct to state that removable dental prostheses, given suitable pretreatment and follow-up regimes, can provide satisfactory solutions . It was Conventional RPD design is frequently bilateral  and consStudies have suggested that at least two teeth on each side should be splinted when extra-coronal distal extension attachment prostheses are used , 12. It The new fabrications techniques for RPD like subtractive CAD-CAM (computer aided design – computer aided manufacturing) or additive SLM (selective laser sintering) and SLS (selective laser melting) are offering promising results –20.The RDP retention includes the selection of the potential abutment teeth taking into the account their prognosis, their position in the arch, as well as the planned prosthesis design. Retainer selection mainly depends on the remaining tooth substance, the intra- and inter-maxillary relationships, esthetics, and financial aspects. The benefits of dental implants as additional retainers are the increased supportive area for the RDP, the minimized load of the soft tissue, as well as the reduced extension of the base of the prosthesis to enhance the patient’s comfort .The aim of this clinical study was to establish the survival of unilateral RPD (u-RPD) compared to bilateral RPD (bi-RPD) with major connector, to assess the overall satisfaction of the patients wearing these dentures as well as their OHRQoL.The study was realized in the Department of Prosthodontics, Faculty of Dentistry, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania during 2016–2021. Thirty-four partial edentate patients were included in the study. Each patient has signed an informed consent in accordance with an Agreement from the Ethical Committee of the University. The study was conducted according to the Declaration of Helsinki.17 patients were treated with u-RPD, and a group of patients with the same size (17) were treated with bi-RPD with a major connector (control group). Both type of dentures had a metal frame and precision attachment: Rhein 83 OT unilateral  and The F.M. hinge , for the u-RPD and external slide attachments, Vario-Soft 3 rod attachment , for the bi-RPD (control group). In the two cases with extended class Kennedy III edentulous areas, on the mesial abutments it was used a resilient  and on the distal abutment a telescopic crown .The patient’s inclusion criteria were: (1) patients without neurological diseases; (2) presenting Kennedy class I, II or extended class III edentate arches, with or without modifications, incorrect treated or not treated; 2. abutment teeth with healthy periodontium or incipient periodontitis (3) the impossibility of realizing implant supported FPD; (4) recalls every six months; (5) direct reline every 12 months? The exclusion criteria were: (1) missing the recalls; (2) missing the reline appointments. (3) patients who do not wear their removable dentures. Following these criteria, the patients were randomly selected.The examined aspects were (a) maintenance of the occlusal contacts and the masticatory function; (b) maintenance of the periodontal health of the abutment teeth; (c) fractures of the removable dentures polymeric structure; (d) fractures of the connectors; (e) chipping of the aesthetic material of the fixed partial dentures and (f) comfort of the patients. The type of the opposite dentition  was also recorded.Periodontal assessment was realized by clinical examination and intraoral radiographic examination. Fractures of RPD’s polymeric structure, fractures of the connectors, chipping of the aesthetic material of the fixed partial dentures were observed during clinical examination.Maintenance of the occlusal contacts were checked by using 40µ articulation paper (Bausch – Germany). Masticatory efficiency was assessed by the administration of a questionnaire (including yes/no questions). The overall satisfaction of the patients was registered on a Likert scale 1 to 5: 1-not satisfied, 2-nearly satisfied, 3 satisfied, 4-very satisfied, 5-exceptional. All the check-ups were realized by two experienced clinicians. The initial periodontal therapy, scaling, professional cleaning and rinsing with chlorhexidine (0.06%%) and peroxide mouthwash (1.5%) was realized in all the cases. After that, the Oral Health Impact Profile-14 (OHIP-14) questionnaire was used to assess the quality of life of the RPD’s wearers included in this study.Strauss&Co, Germany) and a vertical prepared finishing line. After 10 days with provisional FPD’s , the prepared abutment teeth were impressed by using a vinylpolysiloxane impression material in one step technique . The impression of the antagonists was taken with the same material in one step and the occlusion was recorded by using a bite impression material . Then, the try-in of the selective laser sintered (SLS) metal framework was performed  in an individual tray fabricated over the FPD. The occlusion registration was realized with occlusal rims. Maximum two artificial teeth were selected when the FPD was fixed on two mesial abutments. Consecutive. the try-in of the u-RPD and FPD was realized. In the final, the FPD was cemented together with the u-RPD by using a glass ionomer cement (Fuji Plus – GC Japan). The occlusal relationships were adjusted before and after the luting, by using 40µ and 10 µ microns thin articulating papers (Bausch – Germany) Fig. .The treatment of the patients with bi-RPD with a major connector (control group) followed the same clinical steps as in the u-RPD treatments. The same materials and instruments were used during the treatment. After the prosthetic treatment has been completed, the patients were instructed: (1) how to insert and remove the RPD; (2) to remove the RPD during night and keep it in a mug with water; (3) how to clean the RPD’s by using cleaning tabs and a dental brush and toothpaste; (4) to come for the recalls every 6 month and to call any time they had a complaint.The short form of the OHIP-14 questionnaire , 23 was The statistical analysis was performed using SPSS version 25.0  and R project packages for statistical computing. Descriptive statistics were presented as mean ± standard deviation for continuous variables or counts with percentage for the categorical ones.p-value of 0.05 was the threshold for the statistical significance.Kaplan–Meier survival analysis was conducted using two criteria of failure as endpoints, i.e., when either a repair was needed or a complete failure occurred, discriminated according to RPD type. In addition, restorations not meeting the failure criterion during the five-year follow-up period were labeled as “censored”. A The patients with u-RPD presented a mean age of 53.59 ± 11.41) years, 95% CI , while the mean age of patients with bi-RPD was 57.94 (± 13.94) years, 95% CI . The data sample’s characteristics are presented in Table 1.41 yearWe described the RPDs of both groups of patients in Table X2(1) = 10.091, p = 0.001). On the contrary, the group of patients receiving u-RPD presented Kennedy class II in proportion of 76.5%, while the patients receiving bi-RPD presented the same class in a significantly lower proportion 29.4% (Pearson chi-square test X2(1) = 7.556, p = 0.005). A small percent of patients presented Kennedy class III in both groups.We observed no significant differences between the general characteristics of the two groups of patients, i.e., both groups included 35.3% of men and 64.7% of women, both groups had the treatment on mandibula in proportion of 76.5% and maxilla in proportion of 23.3%. The group of patients receiving u-RPD presented Kennedy class I in proportion of 11.8%, while the patients receiving bi-RPD presented the same class in a significantly higher proportion 64.7%  and 4.882 ± 0.078, 95% CI , for the u-RPD and the bi-RPD, respectively  = 0.301, p = 0.584).The five-year survival rates for the two dentures were 94.1% for u-RPD vs. 88.2% for bi-RPD with a major connector, without a statistically significant difference between them  and the bi-RPD’s (84.2%). According to studies recently published, similar survival rates (90- 94.7%) during a 10-year follow-up period were found , 33.The bi-RPD’s were previously compared to u-RPD’s  and the After comprehensive consideration, it is appropriate to choose two abutment teeth for restoration . To protA recent, very interesting three-dimensional finite element analysis  comparedEnding the denture teeth at the mesial cusp of second molar and positioning the occlusal contacts over the ridge crest adequately stabilize the abutment tooth and denture base of u-RPD . All theAnother problem mentioned in the literature is the unnoticed swallow of the u-RPD , but alsAny prosthodontist is aware that treating seniors with removable dentures is a real challenge. Excellent communication with the patient and his family was extremely important during and following the prosthodontic treatment. Further clinical studies on many patients, comparing the u-RPD with and without a distal abutment , using the new technologies would be interesting for restoring seniors’ dentition and for assuring them a better life comfort. The limitations of this study were the small number of patients included, and the comparisons between only two groups of removable denture wearers.Patients receiving u-RPD presented higher levels of treatment satisfaction and better oral health than patients receiving bi-RPD. The survival rates of the treatments u-RPD and bi-RPD were similar.


In the present report, we evaluated adrenergic mechanisms of generalized spike-wave epileptic discharges (SWDs), which are the encephalographic hallmarks of idiopathic generalized epilepsies. SWDs link to a hyper-synchronization in the thalamocortical neuronal activity. We unclosed some alpha2-adrenergic mechanisms of sedation and provocation of SWDs in rats with spontaneous spike-wave epilepsy (WAG/Rij and Wistar) and in control non-epileptic rats (NEW) of both sexes. Dexmedetomidine (Dex) was a highly selective alpha-2 agonist . Injections of Dex did not elicit de novo SWDs in non-epileptic rats. Dex can be used to disclose the latent form of spike-wave epilepsy. Subjects with long-lasting SWDs at baseline were at high risk of absence status after activation of alpha2- adrenergic receptors. We create the concept of alpha1- and alpha2-ARs regulation of SWDs via modulation of thalamocortical network activity. Dex induced the specific abnormal state favorable for SWDs—“alpha2 wakefulness”. Dex is regularly used in clinical practice. EEG examination in patients using low doses of Dex might help to diagnose the latent forms of absence epilepsy . It is widely accepted that the brain’s noradrenergic system regulates working memory, attention function and arousal level ,2,3. It The center of the brain’s noradrenergic system lies in the Locus Coeruleus (LC), which sends wide connections throughout the brain . It contThere are three main types of adrenergic receptors (ARs): alpha1, alpha2 and beta ,27,28. Tsedative, anxiolytic, analgesic, sympatholytic, and opioid-sparing properties and induces a unique sedative response which shows an easy transition from sleep to wakefulness”. Less attention is given to Dex  promoting SWDs. Rats with spontaneous SWDs showed an elevation of spike-wave activity after the administration of Dex (Dexmedetomidine (Dex) is a highly selective alpha2-ARs agonist used in clinical and veterinary practice as a sedative pharmacological agent ,34,35,36n of Dex . Administration of Dex in a very high dose led to a decrease in the number of SWDs. Biphasic enhancement of spike-wave epileptic activity in WAG/Rij rats after Dex injection has recently been reported ,23. A biIn WAG/Rij rats, SWDs mainly occur during the transition from wakefulness to sleep ,40. DuriFurther influence of brain sleep-promoting systems leads to the predominance of slow-wave (delta) oscillations in the cortico-thalamo-cortical network. a pro-longed, generalized, and nonconvulsive seizure recurrent, unprovoked episodes of typical absence seizures” [“acute symptomatic epilepsy as a reaction to the drug” [The agonist of alpha2-ARs, clonidine, systemically injected in the WAG/Rij rat model of absence epilepsy in a very low dose (0.00625 mg/kg) caused prolonged and recurrent spike-wave seizures . The agoeizures” . Pharmache drug” . Here wehe drug” ,48,49,50We performed in vivo pharmaco-EEG investigation in 26 adult rats. All of the rats were bred and raised at our Institute, and they were all intact and not genetically modified. We had 11 WAG/Rij rats  and 1 Wistar rat . The WAG/Rij with spontaneous SWDs and Wistar rats are the most commonly used rat strains in research ,51,52,53The control group consisted of age-matched rats. The inclusion criterion for the non-epileptic group: the number of genuine SWDs in baseline was less than 0.25 per hour (1 SWD per 4 h). The exclusion criterion: the number of genuine SWDs in baseline was higher than 3 per hour. In total, 13 subjects of the NEW substrain were included; subject M5 of the NEW substrain met the exclusion criterion and was moved to the epileptic group .®, Orion Pharma, Espoo, Finland). The estral phase was examined in female subjects. The phases of the estral cycle did not affect the parameters of SWDs.To quickly and safely reduce the number of rats used for testing, rats were used for multiple tests with 14–21 days intervals between tests. Therefore, 1–3 baseline EEGs and 1–3 pharmaco-EEG tests were performed in each rat. A baseline recording preceded each pharmaco-EEG recording. Dexmedetomidine (Dex) was i.p. injected in doses 0.003–0.049 mg/kg (from 0.01 to 0.12 mL of DexdomitorThe density of SWDs (dSWD) was computed as the duration of SWDs in seconds per minute for 5 min and 1 h bins.The number of SWDs (nSWD) per 1 h.The mean duration of SWDs (durSWD) per 1 h.SWDs were detected when wavelet power at 8–10 Hz and 17–20 Hz exceeded individually chosen threshold values . The thrTo further analyse Dex effects in the epileptic group for each animal, we computed the difference of dSWD between baseline and Dex conditions .This part of the study was conducted in 13 non-epileptic control rats of both sexes . Therefore, Dex did not increase genuine SWDs in non-epileptic control rats.I.p. injections of Dex in doses 0.004–0.034 mg/kg .1 rarelyTwo rats (F5 & F6 or 15% of the control group) showed no SWDs at baseline, but some SWDs occurred after Dex injection at dose 0.008 mg/kg . These sNoteworthy is that Dex (0.004–0.034 mg/kg) did not provoke genuine SWDs in non-epileptic subjects but elicited epileptiform activity in the form of irregular spikes, 4–8 Hz sharp waves and occasional spike-wave complexes . The powThis part of the study was performed in 13 rats of both sexes , with the epileptic phenotype confirmed by EEG investigation. This group included 11 WAG/Rij rats, 1 NEW rat and 1 Wistar rat , in whicInjections of Dex in low doses induced long-lasting SWDs b, which The number of SWDs (nSWD), SWDs density  and the mean duration of SWDs (durSWD) were computed. All these parameters, e.g., nSWD, dSWDs and durSWDs, were statistically analyzed with bin = 5 min and 1 h. In addition, we computed the difference in dSWD values between baseline and Dex conditions in 5 min bins for each rat.n = 7 rats, Dex dose 0.003−0.049 mg/kg); (2) ‘Pro-absence effect’—9 rats, Dex dose 0.003−0.037 mg/kg; (3) ‘Severe absence, epi-status (early)’—2 rats; Dex dose 0.008−0.012 mg/kg; (4) ‘Severe absence, epi-status (late)’—1 rat with dose two doses of Dex—0.004 and 0.008 mg/kg. To organize the observed data in meaningful structures, we performed a K-means cluster analysis of SWDs density . For each 5 min bin, we computed the difference between dSWD measured after Dex injection and in the baseline. The resulting differential SWD scores underwent K-means cluster analysis. We used 50% of the time spent with SWDs as an objective EEG-based criterion for status epilepticus. Four clusters of Dex-induced changes in SWDs density were defined b: (1) ‘LLow effect (Clusters 1);Pro-absence (Clusters 2);Severe epilepsy (Clusters 3 and 4).Cluster analysis  showed tp > 0.05). The RM ANOVA indicated that injections of Dex caused an increase in the density of SWDs compared to baseline . In addition, post-hoc analysis  of the data showed that the density of SWDs was significantly higher than in baseline during 1–5 h after injection. The individual results of SWDs density, which are summarized in The variability of Dex-induced effects on SWDs might be explained by individual factors, such as parameters of SWDs at baseline.p = 1.7 × 10−9, p = 2.3 × 10−3) and higher than in rats with ‘low effect’ .SWDs properties were retrospectively analyzed at baseline considering Dex-induced effects on SWDs: ‘low effect’, ‘pro-absence effect’ and ‘severe epilepsy’ b. Properp = 0.18, Surprisingly, the number of baseline SWDs did not differ in different categories of Dex-induced effects  and longer than in rats with ‘low effect’ .Duration of baseline SWDs also linked to Dex-induced effects, as was shown by the significant effect of the ‘category’ : (1) wakefulness , (2) sloI.p. injections of Dex usually provoked sedation characterized by specific EEG and behavioral correlates —light seThe following measures of behavioral/EEG states were analyzed .The latency of the first SWDs after the injection (L1swd).The latency and the total duration of the sedative state The duration of the deep sedation phase .The duration of the first and the second periods of enhanced spike-wave epileptic activity .Injections of Dex elicited two types of EEG/behavioral responses.p < 0.01).The type 1 response  was charThe type 2 response to Dex injection  was charn = 8, r = 0.79, p = 0.019, n = 8, r = 0.94, p = 0.001, n = 8, r = 0.71, p = 0.045, Pearson correlation test was used to determine the dose-specific effect of Dex on the parameters of behavioral/EEG states. Positive correlations were found between the total duration of the Dex-induced sedative state and the dosage of Dex  elicited epileptiform activity in the form of irregular spikes, 4–8 Hz sharp waves and occasional spike-wave complexes in non-epileptic  and in eWe found that systemic administration of Dex  increased the density of SWDs during 1–5 h after injection. Duration of Dex-induced elevation of SWDs could be accounted for by the pharmacokinetics of Dex in rats: the half-life period, T½, is about 1 h . The obsIn 23% of tests in rats with spontaneous SWDs (3 out of 11 rats), i.p. injections of Dex with caused the most severe pro-epileptic effect a median dose of 0.008 mg/kg. These rats showed the longest SWDs at baseline (median 6.00 s).A mild pro-epileptic effect of Dex was found in about 46% of rats. These rats had shorter SWD durations at baseline (median 4.80 s) than rats from the previous group.A low effect of Dex on SWDs was found in the rest, 31% of rats. These rats had the shortest SWDs at baseline (median 4.05 s).SWD-promoting effect of Dex was mostly linked to the epileptic phenotype of rats with spontaneous SWDs, but seemed not to be dose-dependent:A pro-epileptic effect of Dex is associated with the increase of SWDs number rather than with an increase in the duration of SWDs.The most severe effect of Dex was observed in rats with longer baseline SWDs. However, the pro-epileptic effect of Dex did not relate to the number of baseline SWDs. Therefore, subjects with long SWDs at baseline were at high risk of absence status after activation of alpha2-ARs.Status epilepticus is a condition resulting either from the failure of the mechanisms responsible for seizure termination or from the initiation of mechanisms which lead to abnormally prolonged seizures” [We found that relatively low doses of Dex  resulted in prolonged and recurrent SWDs in 23% of epileptic rats. Severe absence epilepsy that lasted longer than 1 h and might be interpreted as epileptic status  with eareizures” . The durAs we have suggested, low Dex doses led to the achievement of alpha2/alpha1 ARs activation ratio, which was favorable for SWDs prolonged enhancement, but were too low to induce the sedative state. This corresponded to the Type 2 response in https://encyclopedia.pub/video/video_detail/628 (accessed on 15 April 2023).In Dex condition, pharmacologically induced genuine SWDs appeared during active wakefulness, but baseline SWDs in WAG/Rij rats usually occurred during drowsiness or slow-wave sleep ,40. ThisThe current study observed two changes in EEG/behavioral states after Dex injections. First, a biphasic increase in spike-wave epileptic activity was associated with entering the sedative state, temporarily suppressing the SWDs. Second, Dex dose-dependently induced light or deep sedation. The deep sedation was associated with the loss of righting reflex (LORR) and stable, strong delta activity in EEG in animals . We did https://encyclopedia.pub/video/video_detail/628 (accessed on 15 April 2023). At the same time, the baseline SWDs in WAG/Rij rats usually occurred during drowsiness or slow-wave sleep [The summary of our results helped add more details to our schema and a pro-epileptic state of “alpha-2 wakefulness” induced by Dex . Accordive sleep . This leBuzsáki et al. 1991 demonstrated that HVS epileptic activity (same as SWDs) in rats could be provoked by activating the specific pool of alpha2 postsynaptic ARs in the thalamus . The remThe lack of sham injections as a control condition is a limitation of the current study. Here we used baseline records for control but not records after control injections. A similar approach was chosen in our previous study on the effect i.p., injections of another alpha2-agonist—clonidine, in which baseline was used as a control condition . In the We have proposed that lower doses of Dex would lead to long and stable spike-wave epileptic activity because the rats were “trapped” in the “alpha2 wakefulness state” favorable for the occurrence of SWDs without entering the sedative state. Nevertheless, a type 2 response to Dex could be observed at the same doses  as a type 1 response . In the current study, different rats had different baseline levels of SWDs and probably different sensitivity to Dex. Therefore a dose-dependent effect considering the type of response was not found. Perhaps a careful investigation of individual dose-dependency could help to define the switch of response type from single phase to biphasic from low to high dose of Dex. Nevertheless, in the type 1 response, the dose-dependent increase of the second phase of enhanced spike-wave epileptic activity duration was demonstrated.The study was conducted on adult WAG/Rij rats and the non-epileptic control “NEW” rat substrain. All rats were bred and maintained at the Institute of Higher Nervous Activity and Neurophysiology of RAS, Moscow (IHNA). The experiments were carried out in accordance with EU Directive 2010/63/EU for animal experiments and were approved by the animal ethics committee of IHNA (protocol No. 4 was approved on 26 October 2021). Rats were kept in environmentally controlled conditions with a 12:12 h light: dark cycle (lights on at 8 a.m.), with constant ventilation and airing. Rats were provided with unlimited access to food and water. Rats were housed in same-sex groups (3–4 subjects per cage) to minimize any potential stressors and to promote social interactions.In total, 26 adult rats of both sexes were used . The average body weight of females was 261 g , and 385 g for males .Chronic electrodes were implanted to record electrical brain activity in free behavior. Epidural electrodes were made of stainless-steel screws , providing recordings of electrical brain activity (EEG). The surgery was performed under isoflurane anesthesia in a stereotaxic apparatus . Three active epidural screw electrodes were implanted over the left/right frontal cortex  and occipital cortex ; the fourth reference screw electrode was placed over the right cerebellum. Coordinates are given in mm relative to the bregma. Care was taken to reduce pain, suffering, and distress as indicated in the experimental protocol approved by the animal ethics committee of IHNA RAS. After the surgery, animals were housed individually under a 12:12 h light: dark cycle (light on 8 a.m.) with free access to food and tap water. The recovery period lasted 10–14 days.Rats were placed in Plexiglas cages (25 cm × 60 cm × 60 cm), and three-channel EEG signals were recorded in freely moving animals under a 12:12 h light: dark cycle (light on at 8 a.m.). Signal inputs were fed into a multi-channel amplifier  via a swivel connector, band-pass filtered between 0.5 and 200 Hz, digitized with 400 samples/second/per channel, and stored in a hard disk. After Dex injections, eFace 1325R video camera  was used to record the rat’s behavior for 2–4 h.No animals were harmed or lost during experiments because Dex was used in therapeutic doses. All rats were born, raised, and given all the same conditions in the same environment. After the surgery, the rats were allowed to recover for ten days and kept in individual cages on the same two lower rows. Experiments were performed in the sound-attenuated room with the same characteristics as in the vivarium. We also performed baseline recording before each Dex injection, which helped reduce some potential biases.® 0.1, Orion Pharma, Espoo, Finland). Dex was injected i.p. in different therapeutic doses ranging from 0.003 to 0.049 mg/kg. To select the most effective dose with the highest pro-epileptic effect, i.e., the central dose, we did pilot experiments in epileptic rats. The effect of Dex was individual, and the doses that were given varied across the central dose. Non-epileptic rats were injected with the same  dose of Dex.All pharmacological tests were done using the agonist of alpha2-adrenoreceptors dexmedetomidine hydrochloride . The preliminary frequency analysis of video-EEG was performed using FTT with window sizes 512, 1024 or 2048 and 50–95.5% window overlap .Visual analysis of video-EEG records was performed 4 h after injection of Dex using LabChart 8 software . The frontal left EEG channel underwent power spectrum analysis , window overlap 50%). Based on raw EEG, EEG power-spectrum and video records (when available), five states were identified: (1) wakefulness, (2) slow-wave sleep, (3) spike-wave activity, (4) deep sedation phase and (5) the state with mixed EEG patterns. To evaluate the whole EEG-behavioral picture of rats’ reaction to Dex administration, video-EEG was recorded in each rat used to assess EEG/behavioral states. We checked whether we could use pure EEG data to identify EEG/behavioral states. The preliminary analysis showed that this approach was reliable.Wakefulness . This stSlow-wave sleep  is charaThe spike-wave epileptic activity  was assoDeep sedation phase . PeriodsState with mixed EEG patterns . PeriodsThe spike-wave complexes are asymmetric, the repetition of spikes within a burst varies from 7.5 to 9.5 Hz with a mean frequency of 8.7 Hz.” However, the majority of pharmacological studies in WAG/Rij rats were done at the University of Catanzaro , and Marmara University  still define SWDs manually . example, ,69,70,71example, ,72. See In the current study, SWDs were automatically detected in the full-length raw EEG using the wavelet-based detection algorithm  lasted, on average, 48–72 h and was characterized by the predominance of leukocytes. Leukocytes were absent in the stage of proestrus and estrus. However, some leukocytes and epithelial cells were detected during the metestrus (6–8 h).Datasets containing the start and end points of automatically detected SWDs were statistically analyzed. First, the densities of SWDs were computed per 5 min as the total duration of SWDs in seconds during 5 min. Second, the number of SWDs, duration of SWDs and density of SWDs were computed per 1 h. Third, the number of SWDs (nSWD), SWDs density  and the mean duration of SWDs (durSWD) were computed with bin = 5 min and 1 h. Finally, we computed the difference in dSWD values for each rat between baseline and Dex conditions with 5 min bins during the first 6 h after injection. These differential dSWD values were further used for K-means cluster analysis.p > 0.05. Next, normally distributed data were analyzed with the repeated measures (RM) ANOVA and post-hoc Duncan test.We used Statistica version 12 to perform statistical analysis of the obtained data. In the first step, all the samples were tested for normality. Next, the normal distribution was assessed with a Kolmogorov-Smirnov test with n < 10), we used non-parametric statistical criteria, such as Friedman ANOVA, Mann Whitney U test, Wilcoxon matched pairs test and Pearson correlation test. The level of significance was set at p < 0.05.For samples that did not show Gaussian distribution or small sample size , which are the encephalographic hallmarks of idiopathic generalized epilepsies. We create the concept of alpha1- and alpha2-ARs regulation of SWDs via NA modulation of cortico-thalamo-cortical network activity. We provided experimental evidence to favour this concept and defined the abnormal state favorable for SWDs to occur—“alpha2 wakefulness”.Our experimental study was performed in rats with spontaneous SWDs (WAG/Rij rats and Wistar) and in non-epileptic control strain NEW. Activation of alpha2-ARs caused severe spike-wave epilepsy up to a status epilepticus in WAG/Rij rats, but not in non-epileptic control. Although in two non-epileptic subjects of NEW substrain, genuine SWDs were recorded after injection of Dex. The NEW substrain has a non-epileptic phenotype, but it has a genetic predisposition to spike-wave epilepsy. Therefore, giving Dex to subjects with a genetic risk for seizures could make them more likely to have seizures.Furthermore, considering that Dex is regularly used in clinical practice, EEG examination in patients using low doses of Dex might help to diagnose the latent forms of absence epilepsy .In epileptic WAG/Rij rats, a pro-epileptic effect of Dex was associated with the increase in the number of SWDs rather than with the increase in duration of SWDs. The most severe impact of Dex was observed in rats with longer baseline SWDs. The pro-epileptic effect of Dex did not relate to the number of baseline SWDs. Relatively low doses of Dex  resulted in prolonged and recurrent SWDs in 23% of epileptic rats that might be interpreted as epileptic status. In all, subjects with long SWDs at baseline were at high risk of absence status after activation of alpha2-adrenoreceptors.We observed two types of changes in the rats’ EEG/behavioral state after the Dex injection. First, a biphasic increase in spike-wave epileptic activity: an initial increase in SWDs soon after Dex injection was followed by the sedative state (when SWDs were suppressed) and then by the secondary increase in SWDs. Second, light or deep sedation dose-dependently induced by Dex.


Cannabis sativa is a plant used for recreational and therapeutic purposes; however, many of the secondary metabolites in the plant have not been thoroughly investigated. Stilbenes are a class of compounds with demonstrated anti-inflammatory and antioxidant properties and are present in cannabis. Many stilbenes present in cannabis have been investigated for their therapeutic effects. Fourteen stilbenes have been identified to be present in cannabis, all of which are structurally dihydrostilbenoids, with half possessing a prenylated moiety. The stilbenes summarized in this analysis show varying degrees of therapeutic benefits ranging from anti-inflammatory, antiviral, and anti-cancer to antioxidant effects. Many of the identified stilbenes have been researched to a limited extent for potential health benefits. In addition, predictive in silico modeling was performed on the fourteen identified cannabis-derived stilbenes. This modeling provides prospective activity, pharmacokinetic, metabolism, and permeability data, setting the groundwork for further investigation into these poorly characterized compounds. Cannabis sativa contains at least 550 different compounds [Cannabis sativa have not been thoroughly investigated, including stilbenes and flavonoids. These compounds, some of which appear to be uniquely found in Cannabis sativa, also have the potential for similar or additional therapeutic benefits, but further characterization is required. The potential benefits of medicinal use of compounds such as stilbenes and flavonoids are evidenced by the therapeutic effects seen in similar compounds to the variants found in cannabis, with anti-cancer, antioxidant, and anti-inflammatory benefits [Current evidence suggests ompounds , with neompounds . The ongompounds . Other sbenefits ,4. OngoiOf the plethora of compounds already identified within cannabis, fourteen have been classified as stilbenes. Stilbene is a chemical constituent with characteristic dibenzal rings connected in either the E or Z configuration around a central double bond A,B that Canniprene  is a pre2 synthase (mPGES) pathways [A major therapeutic effect that canniprene demonstrates is the anti-inflammatory nature of the compound. The investigation of canniprene as an anti-inflammatory stems from prior research into the related polyphenol structures of cannflavin A and B, which demonstrated inhibition of the 5-lipoxygenase (5-LO) and microsomal prostaglandin Epathways . This fopathways  measuredpathways . Similarpathways . Canniprpathways . Inhibitpathways .2 production during inflammation, with mPEGS-1 catalyzing the final step in the biosynthesis of prostaglandin E2 from arachidonic acid [The mPEGS pathway is responsible for a large quantity of prostaglandin Enic acid . Where tnic acid . Canniprnic acid .The relationship between canniprene and cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) was investigated using in silico modeling by Romero Salamanca and Castañeda Castellanos . The inhThe capability of canniprene to act as an anti-cancer agent was investigated by Guo et al. , who tesThe COVID-19 pandemic has brought into focus the severe consequences of a lack of antiviral therapies for diseases such as COVID-19. The SARS-CoV-2 virus has necessitated the investigation of a diverse range of possible treatments and preventative measures to end the pandemic, including phytoligands in cannabis. Canniprene is one of 29 phytoligands investigated by Khattab and Teleb  using inAnother stilbene found in cannabis is cannabistilbene. This compound is present in three forms: cannabistilbene I, cannabistilbene IIa, and cannabistilbene IIb. Of the three structures, only cannabistilbene I is prenylated. There is limited research into cannabistilbene since its initial isolation almost forty years ago by ElSohly et al. , who extThe biosynthesis of cannabistilbene I and II follow different pathways, as made apparent by the prenylation of I but not IIa or IIb. Evidence suggests cannabistilbene I is derived from another stilbene in cannabis, dihydroresveratrol . From diThe same in silico modeling that was performed in canniprene was also investigated with the three forms of cannabistilbene to determine the inhibitory effects of the compounds on COX1 and COX2 . For theThe next prenylated stilbene found in cannabis, 3,4′-dihydroxy-5,3′-dimethoxy-5′-isoprenylbibenzyl , also haRomero Salamanca and Castañeda Castellanos  also invα,α′-dihydro-3′,4,5′-trihydroxy-4′-methoxy-3-isopentenylstilbene (HM1), α,α′-dihy- dro-3,4′,5-trihydroxy-4-methoxy-2,6-diisopentenylstilbene (HM2),and α,α′-dihydro-3′, 4,5′-trihydroxy-4′-methoxy-2′,3-diisopentenylstilbene (HM3)  were firThe potential benefits of HM1, HM2, and HM3 for treating different cancers were tested in vitro by Guo et al. . The celIn addition to testing the efficacy of HM1, HM2, and HM3 as anti-cancer agents, Guo et al.  tested tThe prenylated dihydrostilbene α,α′-dihydro-3,4′,5-trihydroxy-4,5′-diisopenten- ylstilbene  was firsFifteen years after α,α′-dihydro-3,4′,5-trihydroxy-4,5′-diisopentenylstilbene was found in liquorice, it was identified in cannabis by Guo et al.  who were−1s−1) allowed for the compound to be classified as a good antioxidant, close to the 100–1000 k (M−1s−1) range seen in very effective antioxidants [Before α,α′-dihydro-3,4′,5-trihydroxy-4,5′-diisopentenylstilbene was identified in cannabis, the compound was tested for its efficacy as an antioxidant. The absolute inhibition of oxidation rate constants for the sample extracted from liquorice was determined . The ratoxidants . The most well-studied stilbene in cannabis is dihydroresveratrol , a dihydDue to the well-accepted anti-inflammatory actions of resveratrol, the potential for dihydroresveratrol to also be anti-inflammatory has been investigated . InterleThe potential for polyphenol compounds to act as antiproliferative agents towards breast cancer cell lines was investigated by Ávila-Gálvez et al. , who aimAcute pancreatitis is an inflammatory disease that causes premature activation of proteolytic zymogens, causing autodigestion of the pancreatic exocrine cells . AlthougFatty acid binding protein 4 (FABP4) is a lipid chaperone that contributes to insulin resistance, with high circulating levels of FABP4 being associated with type II diabetes . DecreasThe non-prenylated dihydrostilbene 3,4′-dihydroxy-5-methoxy bibenzyl  was firsLipopolysaccharide-induced macrophages can simulate an inflammatory environment in vitro. Su et al.  used thiThe effects that 3,4′-dihydroxy-5-methoxy bibenzyl has on estrogen production have been investigated in animal models. The cannabis plant has established estrogenic effects, and Wirth et al.  aimed toThe compound 3,3′-dihydroxy-5,4′-dimethoxy bibenzyl (gigantol) is a non-prenylated stilbene having been determined in cannabis A. It wasThe gigantol found in cannabis has been demonstrated to have anti-inflammatory effects. Using in silico modeling, the interaction between gigantol and COX1 and COX2 was investigated . Giganto50 of 12.29 μM to 2.92 μM for MDA-MB-468 and 22.66 μM to 8.36 μM for MCF-7 with the addition of 60 μM gigantol to the Cisplatin [The gigantol isomer found in cannabis has also demonstrated anti-cancer effects. Gigantol has been shown to decrease the proliferation of breast cancer cells, including the cell lines MDA-MB-468 and MCF-7, in a concentration-dependent manner . The abiisplatin . Gigantoisplatin . The effisplatin ,65. Gigaisplatin . Gigantoisplatin . These rThe synthetic radical DPPH is used as a model for determining the ability of a compound to scavenge for free radicals due to the visible color change that can be measured with a spectrophotometer. Hydroxyl radicals, on the other hand, are the most toxic naturally occurring radicals, and the ability of a compound to scavenge them can also be measured. Ahammed et al.  used botHernández-Romero et al.  were priThe production of progesterone occurs in the Leydig cells of the male testes, where the sex hormone is derived from cholesterol, and the production can be impacted by gigantol . The strTrypanosomatid parasites are the cause of the disease leishmaniasis, which can be transmitted to humans through the bites of sandflies found in subtropical regions of the world . LeishmaCombretum caffrum. They further elucidated the structure using NMR and then confirmed it through the synthesis of the compound. Combretastatin B-2 is not well characterized in comparison to other related compounds, including combretastatin A-1, A-2, A-3, A-4, and B-1, which have more extensive research and proven health benefits. Although combretastatin B-2 was first identified in 1987, it was not until over thirty years later that it was identified in cannabis by Guo et al. [Combretum psidioides, a different tree in the same genus Combretum, where it was found to be present in an extract [The non-prenylated stilbene combretastatin B-2  was firso et al. . Combret extract . Althoug extract .Guo et al.  investigCannabis sativa [The compound 3-hydroxy-5,4′-dimethoxybibenzyl (3-O-methylbatatasin)  was firss sativa . Marti es sativa  investigs sativa . The inds sativa . Furthers sativa ,78,79. TAs summarized in previous sections, stilbenes found in cannabis have a plethora of potential therapeutic benefits, but more research is needed to understand the extent to which these stilbenes could be beneficial, including their mechanisms of action in the body. In order to further our understanding of these stilbenes and their metabolism, we performed in silico modeling of the compounds and summarized the relevant results. Predictive modeling is beneficial for determining possible drug–drug interactions, substrates and inhibitors of metabolic enzymes, and pharmacokinetic and pharmacological properties, amongst other outcome measures. Modeling was carried out using the ADMET Predictor™ 9.5 . The chemical structures for the fourteen cannabis-derived stilbenes were researched and then drawn using ChemDraw. The compounds were saved from ChemDraw and uploaded as SDF files into ADMET Predictor™ 9.5. The default settings for the metabolism, transporter, and PhysChem modules were used to classify the stilbenes as substrates and inhibitors of various proteins, including human cytochrome P450 (CYP) isoforms, and to predict activity, pharmacokinetic, and permeability data for the stilbenes. Pharmacokinetic data generated were based on the predictive modeling of a 10 mg immediate-release tablet and human liver microsomal clearance. Data outputs from these modules were imported into Excel for collation and analysis. It is important to note that predictions made by ADMET Predictor™ 9.5 have an inherent variability due to the use of mathematical models and assumptions. This should be taken into account when interpreting the results of the simulations.The ability of the cannabis-derived stilbenes to interact with CYP family enzymes in the liver is crucial for understanding both the metabolic pathways the stilbenes follow and the potential interactions they may have with drugs. As summarized in The breast cancer resistance protein (BCRP) is a crucial transporter for the absorption, distribution, and elimination of drugs. In healthy tissue, BCRP is expressed in the intestinal epithelium, liver hepatocytes, and renal proximal tubular cells . BCRP isDespite major progress in treating human immunodeficiency virus (HIV) with antiretroviral therapies, the disease still represents a challenge to the medical and scientific community. Multiple integrase strand transfer inhibitors have been approved as a therapy for HIV due to their ability to prevent HIV-1 replication . HIV-1 iDetermining the percentage of a compound that remains unbound from blood plasma proteins may be beneficial for understanding the efficacy of drug treatment. The unbound drug is considered active and free to be distributed into the target tissues of the body. Our in silico predictions, seen in Understanding and predicting pharmacokinetic properties of these compounds is beneficial to the future pharmaceutical development of these products. In silico modeling also allows for predictions regarding the permeability of the stilbenes to membranes such as the skin, the jejunum, or the blood–brain barrier, as seen in Cannabis sativa so far, though with the number of compounds identified in the plant ever increasing, more are likely to be characterized over time. The fourteen stilbenes have various degrees of investigational research into their therapeutic potential, with some members, such as dihydroresveratrol and gigantol, being very well characterized due to their presence in other plant sources, while others have just recently been discovered. These compounds show promise as therapeutic agents that could be developed into novel medications for a variety of conditions with further research and development. Our in silico modeling of stilbenes will help guide future research into the pharmacokinetic, pharmacodynamic, and therapeutic potential of the compounds. Further research must be conducted regarding poorly characterized stilbenes and yet uncharacterized compounds to be discovered in Cannabis sativa to fully understand the therapeutic potential of these polyphenols.In conclusion, fourteen bibenzyl stilbenes have been identified in


Sunburn is caused by prolonged exposure to ultraviolet (UV) rays from the sun, resulting in redness of the skin as well as tenderness, swelling, and blistering issues. During the healing process, it can cause peeling, irritation, and some long-term effects, including premature aging, pigmentation, and a high risk of skin cancer. Rutin has antioxidant and anti-inflammatory effects, which could potentially reduce inflammation and soothe sunburned skin. The objective of the current proposal is to develop and create carbopol gel-encased glycerosomes for the treatment of sunburn. The Design of Expert (DoE) technique was used to optimize the proposed formulation and was subjected to various characterization parameters such as nanovesicles size, polydispersity index (PDI), surface charge, entrapment efficiency (EE), and surface morphology. The optimized rutin-loaded glycerosomes (opt-RUT-loaded-GMs) were further characterised for drug release, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, and confocal laser scanning microscopy (CLSM). The formulation showed sustained release, greater permeation into the skin, and good antioxidant activity. The dermatokinetic study of opt-RUT-loaded-GMs confirms that the Rutin hydrate had better retention in the epidermis as compared to the dermis, owing to its potential for long lasting protection after topical application. It was observed that the prepared formulation was stable, highly safe, and had good sun protection factor (SPF) values that could be used as a suitable option for topical drug administration to maximize the therapeutic efficacy of the drugs. The skin, being the largest and most exposed organ of the body, is also highly vulnerable to photodamage resulting from sunlight exposure and other environmental factors. Although it plays a vital role in the production of vitamin D and is beneficial in the treatment of conditions such as psoriasis, dermatitis, and jaundice, prolonged exposure to ultraviolet (UV) radiation poses significant hazards. Chronic UV radiation exposure can lead to various adverse effects, including sunburn, erythema, inflammation, photoaging, and increased radiation levels. Among the different types of UV rays, ultraviolet-A (UV-A 320–400 nm) and ultraviolet-B (UV-B 290–320 nm) rays are particularly responsible for causing skin damage. While the ozone layer provides protection against the most harmful ultraviolet-C UV-C 100–280 nm) rays, UV-B (over 5%) and UVA (approximately 90–95%) radiation penetrate through to the Earth’s surface, thereby impacting ecosystems and human well-being. Consequently, exposure to UV radiation can exert a significant influence on human health . Th. Th16]. The antioxidant potential of rutin hydrate was compared to that of a standard sample (ascorbic acid) The Opt-RUT-Loaded-GMs gel formulation had a homogeneous texture and appearance, was free of any discernible lumps, and had a smooth consistency. The pH of the gel formulation was observed in the range of 5.6 ± 0.15, whereas viscosity was found out to be 1566.667 ± 78.80 cp. The hardness, consistency, cohesiveness, and work of cohesion values for the gel were found to be 255.94 g and 291.64 g. s, 200.39 g, and 248.29 g. s, respectively . The extThe findings of an in vitro release study of opt-RUT-loaded-GMs gel versus RUT suspension are shown in 2 value of the Higuchi model was found to be close to 1 and highest among all kinetic models confirming that it is best fit model. The results showed that the opt-RUT-loaded-GMs gel followed the Higuchi kinetic model (R2 = 0.9472), [The release kinetics were investigated by putting the data into various kinetic model equations. 0.9472), . Whereas0.9472), .Four groups of SPF values are distinguished: non-sunscreen, low, medium, and high. If the score is less than 2, it is not in the category of sunscreens (non-sunscreen); if it is between 2 and 11, it offers only little protection (low SPF); if it is between 12 and 30, it offers medium protection (medium SPF); and if it is greater than or equal to 30, it offers strong protection (high SPF) . The resAccording to confocal laser scanning microscopy, the hydroalcoholic solution of Rhodamine B dye was only able to reach the skin’s outermost layers with a permeation upto 5 µm A. RhodamSkin max and AUC0–8 values observed in both layers upon application of the opt-RUT-loaded-GMs gel. This improvement can be attributed to the nanosized vesicles present in the gel, which facilitate penetration into the skin’s lipid layers. Several, dermatokinetic parameters of opt-RUT-loaded-GMs gel and RUT conventional gel are listed in According to the results of the dermatokinetic study, as shown in The results of the skin irritation test are displayed in Opt-RUT-loaded-GMs display phase dispersion and particle aggregation, leading to an increase in sedimentation rate, following a storage period of three months at a temperature of 40 °C. At the conclusion of a three-month period, the vesicles of the GMs exhibited an increase in size to a measurement of 251.26 ± 2.31 nm. The phenomenon of improved particle sizes can be attributed to the conglomeration of vesicles, thereby elevating the likelihood of phase separation and sedimentation. The entrapment efficiency of drugs was reduced to 69.06 ± 0.448 due to the leakage from the GMs. Based on the empirical findings, it can be concluded that the optimal storage temperature for Opt-RUT-loaded-GMs is 4 °C. Whereas, opt-RUT-loaded-GMs gel formulation was found to be stable for 3 months at 4 ± 2 °C and 40 ± 2 °C/75 ± 5% RH.A glycerosomal gel formulation containing rutin hydrate was effectively developed, optimized, and characterized. The predicted values in BBD exhibited a positive correlation with the observed values. The particle size of the formed Opt-RUT-loaded-GMs fell within the appropriate range for the intended use of topical delivery. The zeta potential analysis demonstrated that the formulation exhibited good stability. The incorporation of the API into the prepared Opt-RUT-loaded-GMs was confirmed by the results obtained from DSC and XRD studies. While the FTIR confirmed the API-excipient compatibility. Glycerosomes provided better penetration in comparison with the suspension. The Opt-RUT-loaded-GMs showed sustained release and good stability. Better penetration of glycerosomal gel than conventional gel was observed in a dermatokinetics study. Furthermore, the formulation was assessed for its potential in treating sunburn, which demonstrated effectiveness in treating sunburn, exhibiting a desirable sun protection factor (SPF) value and displaying favorable antioxidant properties. The formulation is determined to be suitable for safe administration via topical delivery as confirmed by a skin irritation study. Results confirmed that the formulated gel has the potential to maintain stability over prolonged periods when stored under normal conditions.® 934P NF  was received as a gift sample from Lubrizol Advanced Materials Inc., . All the other solvents such as methanol, chloroform, ethanol, and octanol used were of laboratory grade, procured from Merck .Rutin Hydrate (RUT) was procured from Sigma-Aldrich, , a product of China. Phospholipid 90G was procured from Lipoid, Ludwigshafen, Germany. Sisco Research Laboratories PVT LTD, Mumbai, India provided the cholesterol, DPPH, and rhodamine-B. Other chemicals like glycerol, HPLC Water, disodium hydrogen phosphate, potassium dihydrogen phosphate and HCL were purchased from SD Fine Chemicals, . CarbopolA Quality Target Product Profile (QTPP) was developed to ensure that the final formulation meets the required standards for safety and efficacy, establishing the foundation for quality by design and ensuring the finished product’s desired performance . The QTPw/v glycerol) was used, and the two phases were mechanically stirred at 40 °C for one hour. The vesicles were then subjected to sonication for half cycle, which equated to 60 s, with a 3 s pause every 15 s. To remove any excess unentrapped drug, the resulting formulation was centrifuged at 7500 rpm for 10 min at 4 °C before being lyophilized for future use [The thin film hydration approach was employed for the preparation of RUT-loaded-GMs . CholestThe utilization of Design of Experiment (DOE) has been implemented in the development of glycerosomes. The optimization of RUT-loaded-GMs was conducted using the Box–Behnken design, which is a statistical experimental design technique that involves three factors with three levels each. The Design-Expert software  was utilized for this purpose. The study aimed to optimize various process parameters, namely Phospholipid 90G (X1), cholesterol (X2), and Glycerol (X3), to assess their impact on the vesicle size (Y1), polydispersity index (Y2), and entrapment efficiency (Y3) of glycerosomes . The indTo measure the vesicle size, polydispersity index (PDI) and zeta potential the dynamic light scattering was employed using the Malvern Zetasizer, Nano ZS, Oxford, UK. Before analyzing the aforementioned parameters, the formulation was diluted 50 times approximately. The scattering angle was set at 90° and the temperature was held constant at about 25 ± 2 °C. Prior to experimentation, distilled water served as a control for the undiluted formulations. The study was conducted in triplicate to ensure reliability ,29.Using the ultracentrifugation method, the entrapment effectiveness of the opt-RUT-loaded-GMs was assessed. A 10 mL glass centrifuge tube was filled with 2 mL of the formulation after the formulating glycerosomal preparation. The prepared formulation was diluted with distilled water to 5 mL and centrifuged  at 2000 rpm for 20 min while using a cooling centrifuge to separate the undissolved drug from the formulation. Ultracentrifugation was then performed for 30 min at 10,000 rpm to isolate the glycerosomes. The supernatant was collected from the pellets after centrifugation, and the amount of unentrapped drug was determined utilizing a UV spectrophotometer at a wavelength of 259 nm (Shimadzu-UV-1601). A UV calibration curve was generated for Rutin, which followed the formula: y = 0.0257x + 0.0358, where x represents absorbance and y represents rutin concentration. The coefficient of determination (R2) for the calibration curve was determined to be 0.99. Rutin content in glycerosomal formulation was determined by dividing the amount of drug entrapped  by the total amount of drug added during formulation preparation [The nature and entrapment of RUT was confirmed by performing Differential Scanning Calorimetery  for drug samples and opt-RUT-loaded-GMs. A small portion of the sample was quickly sealed in an aluminum pan. The instrument was adjusted to a temperature range of 40 to 400 °C. The samples were heated at 10 °C/min using a nitrogen flow of 60 mL/min. 5% of mannitol was added as a cryoprotectant prior to freeze-drying the opt-RUT-loaded-GMs formulation for lyophilization .−1 [An FT-IR spectrometer was used to obtain FT-IR spectra . Potassium bromide was mixed separately with the pure API-RUT, blank opt-GMs formulation containing all excipients such as Phospholipon 90G, glycerine and cholesterol, and opt-RUT-loaded-GMs formulation (after lyophilization). The powders were compressed at a pressure of 15 tons for 10 min in a hydraulic press to create the potassium bromide discs and scanned from 4000 to 400 cm−1 .−1 was used to take the measurements. Rutin hydrate and opt-RUT-loaded-GMs that had been lyophilized had their XRD patterns identified utilizing the Origin software [An X-ray diffractometer  was used to assess the drug’s encapsulation with a scattering angle range of 20–80°. The diffractometer functioned at a voltage and current of 40 kV and 40 mA, respectively, and a rate of 0.6 minsoftware .Transmission electron microscopy (CRYO-TEM (TALOS S), Thermo Sceintific, Waltham, MA, USA.) was utilized to assess the surface morphology of the opt-RUT-loaded-GMs. A drop of prepared vesicular solution was placed on a carbon-copper coated grid and 1% phosphotungstic acid was used to negatively stain the sample. To prevent evaporation, the vesicular solution was applied to a carbon-coated TEM grid for 10 min in a moist environment, then, the surplus liquid was drained with filter paper. The prepared samples were observed using Transmission Electron Microscope and images for vesicular nanoparticles were captured .The standard 2,2-diphenyl-1-picrylhydrazyl (DPPH) technique was used to assess the antioxidant potential of RUT. The DPPH free radical method is widely used to evaluate a compound’s antioxidant potential. The electron-donating ability of antioxidants causes the violet-colored DPPH solution to turn colorless at room temperature. The standard in this assay was ascorbic acid . The sample (0.5 mL) was first dissolved in 3 mL of methanol before being treated with 0.3 mL of DPPH methanolic solution. The resulting reaction mixture was allowed to continue reacting for an additional hour in a dark environment. The color change indicates the sample’s antioxidant potential as a result of its hydrogen-donating ability. The blank contained 3.3 mL of methanol and 0.3 mL of sample, whereas the control contained 3.5 mL of methanol and DPPH solution (0.3 mL). At 517 nm, the sample was spectrophotometrically (Shimadzu-UV-1601) examined.A graph showing the percentage of DPPH scavenging activity vs. different RUT concentrations was used to calculate the IC50 value for API (concentration at which it shows 50% of antioxidant activity) .® 934P NF . was utilized for preparing gel formulations. The required carbopol® powder (1%) was dissolved in distilled water and was kept overnight for proper mixing. The next day, 2–4 drops of triethanolamine were added to the carbopol® mixture with continuous stirring, which led to the formation of a gel base due to the alteration in pH. Then, prepared opt-RUT-loaded-GMs were mixed with the gel base under continuous gentle stirring to prevent any bubble formation. Then, gel samples were continuously stirred for 60 min on a magnetic stirrer and opt-RUT-loaded-GMs Carbopol® gel was obtained [The gelling ingredient carbopolobtained .One gram of Opt-RUT-loaded-GMs gel formulation was weighed in a 50 mL volumetric flask and diluted with double distilled water and left for 2 h. The pH of the gel formulation was measured using a glass microelectrode  by keeping it in contact with the diluted gel for 1 min. Three distinct batches of gels were tested.All the gel formulations were transferred in transparent containers and were visually inspected for homogeneity. It was examined for the appearance of any type of aggregates in the gel formulation . The visn = 3), and the average time of the three trials was used in subsequent calculations.The Opt-RUT-loaded-GMs gel spreadability was assessed utilizing the parallel plate technique. In this experimental protocol, a circular area with a diameter of 1 cm was marked on a glass plate, and 500 mg of the formulated gel was subsequently applied onto this area. Another glass plate was then placed on top of the gel. A duration of 5 min was allocated for the placement of a weight measuring 500 g onto the upper plate of the glass. The expansion of the diameter was observed as a result of the spreading of the gel . The entS is spreadability (g/s), M is the weight attached to top slides (gm), and T is time (s).The following formula was used to determine the spreadability.n = 3 [To evaluate the formulation’s extrudability, a closed, collapsible tube’s crimped end was forcibly squeezed utilizing weights. The formulation was extruded once the cap was taken off until the pressure subsided. The weight in grams required to extrude a 0.5 cm ribbon of the gel formulation in 10 s was calculated. The results were made in regard to the average extrusion pressure in grams i.e., n = 3 .The gel was examined using a back extruding probe of 30 nm diameter in a texture analyzer . The consistency firmness, cohesiveness, consistency, and viscosity index were all determined using this technique .n = 3).The in vitro release study of opt-RUT-loaded-GMs gel was carried out using a dialysis method. The pre-activated dialysis bag (MW-12 kDa) was utilized, both opt-RUT-loaded-GMs and RUT suspension was added to the bag following both the ends were tied using sealing clips. Phosphate buffer saline  was used as dissolution media to immerse the dialysis bags in a 500 mL beaker. The beaker was then placed on a magnetic stirrer maintained at a temperature of 37.5 ± 0.5 °C and a moving speed of 100 rpm. A 1 mL sample aliquots were obtained at 0.5, 1, 2, 4, 6, 8, 12, and 24-h intervals. In order to equalize the sink conditions, the receiver compartment received 1 mL of fresh dissolution medium. The drug content of the withdrew samples were measured using UV spectrophotometer (Shimadzu-UV-1601) at 259 nm following dilutions . The druThe UV-VIS spectrophotometry was used to determine the effectiveness of RUT as sunscreen prepared into opt-RUT-loaded-GMs gel formulation and RUT suspension. The gel formulation and suspension were divided into different concentrations, F1 = 0.02%, F2 = 0.05%, and F3 = 0.1%. Each concentration was measured at intervals of 5 nm between 290 and 320 nm utilizing a UV-spectrophotometer (Shimadzu-UV-1601). Distilled water was utilized as a blank. The SPF value was determined using the absorbance value obtained for samples at different concentrations .SPF = CF × CF = Correction factor (10)EE = Erythmogenic effect of radiation with wavelength λI = Solar Ray Simulation SpectrumAbs = spectrophotometric absorbance values at wavelength λ.w/v Rhodamine B dye and hydroethanolic solution of Rhodamine B dye was applied to rat skin in a non-occlusive manner fixed on Franz diffusion cell . The skin was maintained at 37.5 ± 0.5 °C with continuous stirring at 100 RPM for 8 h. The treated skin was taken out of the diffusion device after 8 h and rinsed with PBS solution to remove any leftover hydroalcoholic solution and adhering formulation. After being longitudinally cut with a microtome, the samples were mounted on glass slides. Confocal Lasser Scanning Microscopy  was used to examine the fluorescent intensity of Rhodamine B dye on the slides. The sample was scanned through the z-axis using an Argon laser with excitation and emission wavelengths of 540 nm and 625 nm, respectively [The study was performed on abdominal skin of rats to determine the penetration potential of GMs gel formulation. The opt-GMs gel formulation containing 0.02% ectively .2). After 0.5, 1, 1.5, 2, 3, 4, 5, 6, and 8 h, the entire skin sample was removed from the assembly. The formulation was adhered to the removed skin samples and was rinsed off three times with normal saline, and then the sample was immersed in warm water (60 °C) for 2–3 min. Following this, the epidermal and dermal layers of the rat’s skin were sliced separately, depauperated with 5 mL of methanol for 24 h at 37 °C, and the drug was completely extracted. The methanol solution was then filtered through a 0.25 µm pore size nylon syringe-driven filter, and the RUT content was determined in triplicate using UV spectroscopy (Shimadzu-UV-1601). Non-compartment dermatokinetics was used to calculate the concentration of RUT in each sample at each time point. PK solver software was used to estimate dermatokinetic parameters such as Cskin-max, AUC0–8 h, Ke, and Tskin-max [n = 3).The goal of the investigation was to determine the drug’s quantitative concentration in rat skin. A dermatokinetic study was conducted to determine the concentration of the drug in the epidermis and dermis layers of rat skin. The analysis was carried out by applying opt-RUT-loaded-GMs gel formulation and RUT suspension to prepared rat skin that had already been fixed on Franz diffusion cells , Jamia Hamdard vide Protocol Number: 2055. The committee is registered with the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) (173/GO/ReBi/S/2000/CCSEA 28 January 2000). The adult Wistar rats weighing in the range of 210–250 g were placed in polypropylene cages in the animal house adhering to IAEC and CPCSEA guidelines.The Draize patch test was employed to investigate the potential irritation at the site of application. The study employed adult Wistar rats as experimental subjects. The dorsal region of the rats was shaved using a electrical clipper and razor. The rats were subjected to a 24-h period of observation to monitor for any injuries that may have been caused. The following day, a gel containing 0.5 g of opt-RUT-loaded-GMs was applied to the shaved area. The negative control group did not undergo any treatment, while the positive control group was treated with a 0.8% formalin solution. Erythema and edema were observed at various time intervals. The Draize scoring system was employed, utilizing a scale of 0 to 4, with 0 denoting the absence of erythema and edema, and 4 indicating the most severe manifestation of erythema and edema .A stability investigation was performed on the optimized RUT-loaded GM liquid formulation and gel formulation to evaluate whether there will be any physical or chemical changes during storage. The formulation was stored for 3 months in a Borosil amber-colored glass container at 40 ± 2 °C, 75 ± 5%RH and 4 ± 0.5 °C for a stability assessment. Following storage, samples were obtained at 0-day, 1 week, 2 weeks, 1 month, 2 months, and 3 months intervals. Various parameters such vesicle size, PDI, % entrapment efficiency, physical appearance, color, phase separation, clarity, pH, homogeneity and washability were then used to determine the stability .


Aeromonas caviae, which co-carrying blaRSA-1 and blaNDM-1 genes. 211703 was isolated from the patient’s cerebrospinal fluid drainage sample in a Chinese tertiary hospital.The objective of this study is to thoroughly analyze the detailed genomic characteristics of clinical strain 211703 of Carbapenemase NDM was detected by the immunocolloidal gold technique. The MIC values were determined by VITEK2. The whole genome sequence of 211703 was analyzed using phylogenetics, genomic comparison, and extensive dissection.blaRSA-1 harbored on integrative and mobilizable element (IME) Tn7413a, and blaNDM-1 harbored on integrative and conjugative element (ICE). The ICE and IME were all carried on the chromosome of 211703 (c211703). Detailed comparison of related IMEs/ICEs showed that they shared similar conserved backbone regions, respectively. Comprehensive annotation revealed that blaRSA-1 was carried by the gene cassette of a novel integron In2148 on Tn7413a, and blaNDM-1 was captured by an insertion sequence ISCR14-like on the ICE of 211703. We speculated that mobile genetic elements (MGEs) such as ICE and IME facilitated the spread of resistance genes such as blaRSA-1 and blaNDM-1.This study revealed that 211703 only contained a single 4.78 Mb chromosome (61.8% GC content), and no plasmids were discovered in 211703. 15 different types of resistant genes were detected in the genome of 211703, including A. caviae 211703, and an in-depth discussion of multiple acquisition methods of drug resistance genes in Aeromonas. To the best of our knowledge, this is the first report of A. caviae carrying blaRSA-1 even both blaRSA-1 and blaNDM-1, and this is the first bacterium carrying blaRSA-1 isolated from the clinical setting.In conclusion, this study provides an overall understanding of the genomic characterization of clinically isolated  Aeromonas comprises a group of Gram-negative bacterium worldwide distributed in aquatic and soil environments  account for the vast majority of human infections and clinical isolates , and cefotaxime . The clone exhibited a β-lactam resistance phenotype typical for Extended Spectrum Beta-Lactamase (ESBL) production and showed a great resistance challenge  is one of the most common carbapenemases in cterales , which c in 2008 . Since the world . In addi in 2018 . Open rehallenge . AlthougattL (attachment site at the left end of the ICE), int (integrase), xis (excisionase), rlx (relaxase), nic/oriT , cpl (coupling protein), a P (TivB)- or F (TivF)-type T4SS machinery (mating pair formation), and attR (attachment site at the right end of the ICE). IMEs are autonomous in integration but nonautonomous in conjugation. IMEs contained no conjugal transfer genes and typically have attL, int, rlx, oriT, and attR.Integrative and conjugative element (ICE)  and inteA. caviae strain 211703 co-carrying blaNDM-1 and blaRSA-1 was identified in clinical setting. According to the sequencing results, the whole genome of A. caviae strain 211703 contained only the chromosome (c211703), and no plasmid was found. All resistance genes including blaNDM-1 and blaRSA-1, and antimicrobial susceptibility profiles of A. caviae 211703 were obtained. The genetic dissection of strain 211703 of A. caviae revealed the presence of ICE carrying the blaNDM-1 gene and IME carrying the blaRSA-1 gene, both of which had integrated into the chromosome.In this work, a carbapenem-resistant A. caviae strain co-carrying blaNDM-1 and blaRSA-1. At the same time, this is the first report of strains carrying blaRSA-1 gene found in a clinical setting. This research will provide a new understanding for the genetic context of novel antibiotic resistance genes in A. caviae, and in-depth insights for the integration, transformation, conjugation, and mobilization of MGEs related to resistance genes under the selection pressure.Comprehensive genomic comparisons of above ICE and IME with their closely related elements were performed, respectively. According to our understanding, this is the first report of A. caviae strain 211703 was isolated from the patient’s cerebrospinal fluid drainage sample initially identified by Vitek 2. Later, bacterial species identification using average nucleotide identity (ANI) analysis (http://www.ezbiocloud.net/tools/ani) based on genome sequences finally proved that 211703 belongs to A. caviae  was admitted to the neurosurgery department, due to sudden unconsciousness and vomiting for three and a half hours. The diagnosis was spontaneous subarachnoid hemorrhage, hemorrhage in the third, fourth and lateral ventricles. After admission, right ventricle drainage was performed. Fluid replacement, brain protection, prevention of bleeding infection, and neurological rehabilitation were given. On the eighth day of admission, the patient was considered intracranial infection with the body temperature 37.9°C and persistent unconsciousness, and a carbapenem-resistant . caviae .The drug minimum inhibitory concentrations (MICs) of 211703 were determined by BioMérieux VITEK2 Table 1.Escherichia coli EC600 being used as a recipient, and the strain 211703 as a donor. The donor and recipient strains were grown in three milliliters (mL) brain heart infusion (BHI) broth overnight at 37°C. And then, 50 μL of donor strain culture was mixed with 500 μL of recipient strain culture (v:v = 1:10) and 4.5 mL of fresh BHI broth. In addition, 100 μL of the mixture was applied onto a cellulose filter membrane  already placed on a BHI agar plate. After incubation at 37°C for 16h-18h, the filter membrane was taken out and vortexed in 1 mL of BHI broth. The vortex mixtures were plated on BHI agar plates containing 2 mg/L imipenem and 1,500 mg/L rifampicin for the selection of the transconjugants. However, repeated conjugation experiments failed to acquire transconjugants.Conjugal transfer experiments were carried out with rifampicin-resistant A. caviae 211703 was isolated using the Gentra Puregene Yeast/Bact. Kit . The TruePrepTM DNA Library Prep Kit V2 and the SQU-LSK109 Ligation Sequencing kit were used for libraries preparation separately. After the preparation of the library was completed, it was separately sequenced on an Illumina HiSeq X Ten platform  and GridION X5 platform . Raw data from the HiSeq X Ten platform and the GridION X5 platform were trimmed to obtain the high-quality clean reads (clean data) by Canu v1.8 (https://canu.readthedocs.io/en/latest/index.html). The paired-end short Illumina reads and the long Nanopore reads were assembled de novo utilizing Unicycler v0.4.8.0 (https://github.com/rrwick/Unicycler).Bacterial genomic DNA of A. caviae sequenced at three levels  were downloaded from NCBI (last accessed on 15 Aug 2022), which were isolated from various sources from 2004 to 2021 .ORFs and pseudogenes of 211703 genome were predicted using RAST 2.0 . FurtherA. caviae 211703 (chromosome c211703) was submitted to the GenBank database, under accession number CP092181. The Illumina reads of A. caviae 211703 was deposited as a SRA in the GenBank database (accession number PRJNA934671).The complete sequence of A. caviae 211703. The results showed that 211703 exhibited resistance to a broad range of tested antimicrobials except aztreonam, amikacin, tigecycline, and minocycline (A. caviae 211703 (arr2 and aphA6), β-lactams , macrolides (mph(A), mph(E), and msr(E)), quinolones (qnrVC1), sulphonamides (sul1), and other antibiotics.Antimicrobial susceptibility tests were performed on ocycline Table 1.A. caviae strains available from GenBank of NCBI whose assembly levels are scaffold, chromosome, or complete, phylogenetic analysis was performed to explore the evolutionary relationships and potential associations  was used as the reference. Strain OnP3.1  was used as the outgroup. After multiple sequence alignment, a total of 394,778 SNPs located in the core genome regions were identified and extracted. Using these SNPs’ dataset, a maximum likelihood (ML) phylogenetic tree was constructed. Based on the genome of 211703 and all ciations . A totalmanually Table S1A. caviae 211703 was obtained by high-throughput sequencing. Genome sequencing result revealed that A. caviae 211703 contained only chromosome  with a length of 4,783,384 bp, and no plasmid was found in its genome after verification  identified on c211703, MLST typing method proved that 211703 belongs to ST928.The whole genome sequence of fication Table 2.blaNDM-1) and one IME  (7413a). In order to explore the similarities and differences in the acquisition of resistance genes and the genetic environment, precise comparisons were performed with related ICEs and IMEs, respectively.Among the 16 resistance locus existing on c211703, 15 of them were concentrated on one ICE  –IS6100 unit carrying mph(A), encoding proteins conferring resistance to macrolides (26–mph(A)–IS6100 unit, IS26 was shared by IS26–mph(E)–IS26 unit followed by a truncated tniA encoding DD(35)E transposase TniA. A pair of 25-bp IRi (inverted repeat at the integrase end) and IRt (inverted repeat at the tni end) flanked In2148. On the outside of IRi and IRt, there were 5-bp DRs , respectively.In2148 was a novel concise class 1 integron containing primary components 5’-conserved region (5’-CS), 3’-conserved region (3’-CS), and a gene cassette array (GCA) crolides . In the 7413a as a reference. BLAST analysis revealed that a total of seven IMEs in the GenBank database were similar to Tn7413a, sharing 100% coverage and >99% identity for gene int from Tn7413a. These seven IMEs were included in this analysis, and the gene structures of these IMEs were detailed annotated manually. Due to their structural similarity to Tn7413a, these seven IMEs were designated as Tn7413b-h as listed in To investigate the structural variations among these IMEs, a BLAST analysis was performed using the backbone region sequences of Tn7413b-h ranged in length from 29,639 bp to 59,408 bp, and were all distributed in Aeromonas. Similar to Tn7413a, the boundaries of these seven IMEs were a pair of 18-bp attL/attR. The backbone regions of Tn7413b-h were almost identical to that of Tn7413a, but differed in the accessory regions –IS6100 unit, which was same as in Tn7413a. For Tn7413c-e and Tn7413h, all contained single or multiple Tn4352, a composite transposon involved in kanamycin resistance –IS26 unit, which was identical to Tn7413a. Tn7413g contained a putative resistance unit ISCR2–floR unit. ISCR elements were bounded by an origin (oriIS) downstream and a terminus (terIS) upstream, responsible for capturing resistance genes –IS26 unit sharing IS26, carrying resistance genes catA2 and tetA(D), respectively.Tn regions Figure 2uF–aadA2 . In Tn74sistance . For Tn7ce genes . In Tn74blaRSA-1, the genetic environment of the previously reported blaRSA-1, which belonged to Aeromonas spp. strain 1402 was also included in this study –IS6100 unit.Furthermore, in order to explore the genetic environment differences of is study . The resA. caviae 211703 was located on chromosome from 3,757,651 bp to 3,865,845 bp with a length of 108,195 bp, bounded by a pair of 19-bp attL/attR  , and tetA(G), which together constituted the end of the blaNDM-1 region from ICE of 211703. There were four ISCR14-like elements upstream of the ISCR14-like captured ΔTn125, which indicated that multiple insertions or recombination occurred.ICE of unctions Figure 3y ISCR1) , the blaA. caviae 211703 (100% coverage and >95% identity for gene int from ICE of A. caviae 211703)  with A. caviae 211703, which indicated that ICE of A. caviae 211703 may be transferred from Pseudomonas to Aeromonas. Only one of the 21 ICEs originated from Aeromonas (ICE of NUITM-VA2). As shown in ars locus, and the backbone region of conjugal transfer, and the differences mainly occurred in the accessory region where the blaNDM-1 region was located.BLAST analysis revealed that a total of 21 ICEs in the GenBank database were almost identical to ICE of  211703) Table S5P. juntendi PP_2463 (sharing the most similar accessory regions with the ICE of A. caviae 211703) and the ICE of A. caviae NUITM-VA2 (the only one ICE from A. caviae), were specially annotated and analyzed. Detailed annotation of their blaNDM-1 region and comparison with blaNDM-1 region from ICE of 211703 were performed (blaNDM-1 region from ICE of PP-2463/NUITM-VA2 shared a common structure in upstream(Δorf5–sul1–ΔqacED1–ISCR14-like) with blaNDM-1 region from ICE of 211703, following by a replacement of gene blaPME-1. The genetic structures identified included an ISCR14-like ΔTn125 carrying the blaNDM-1 gene, as well as a ΔISCR14-like–blaPME-1–ISCR14-like structure, which was not present in the ICE carrying blaNDM-1 in strain 211703. The only difference found between the blaNDM-1 regions from ICE of PP-2463 and NUITM-VA2 was an insertion of aphA6–ISCR14-like, which was also present in the blaNDM-1 region from ICE of 211703. Despite this variation, all three blaNDM-1 regions shared a common structure downstream, consisting of floR–tetR(G)–tetA(G). However, there was an additional insertion of the ISStes1 sequence downstream of the blaNDM-1 regions in both ICE of PP-2463 and NUITM-VA2.Among the 21 ICEs, the ICE of Aeromonas has received more attention since the first isolate from France harboring an acquired blaIMP gene was reported in 2007  that are similar in structure to the IME (Tn7413a) where the integron In2148 carrying blaRSA-1 is located , and was captured by ISCR14-like rather than the common ISCR1, and was not further integrated into the integron. In addition, more than one insertion and recombination of ISCR14-like related elements occurred in the blaNDM-1 region from ICE of 211703, and similar phenomenon also appeared in the blaNDM-1 regions of Pseudomonas PP-2463 and Aeromonas NUITM-VA2. The blaNDM-1 regions from 211703, PP-2463, and NUITM-VA2 showed high homology, indicating that such blaNDM-1 environments have existed across species. This new blaNDM-1 genetic environment may herald a new way of recombination, which needs to be focused on. Further, the similar related ICE included in this study are mainly derived from Pseudomonas , and a high degree of structural similarity was shared by blaNDM-1 regions from both A. caviae 211703 and Pseudomonas PP-2463. This may indicate that the ICE in A. caviae 211703 was transferred from Pseudomonas.he world . In Aeror or two . In the A. caviae strain harboring both blaRSA-1 and blaNDM-1 isolated from the clinical patient. To the best of our knowledge, this is the first report of A. caviae harboring blaRSA-1 even both blaRSA-1 and blaNDM-1 from clinical setting in the world. For the first time, we detailed annotated the integron where the blaRSA-1 gene is located and deeply analyzed its related genetic environment. Further, the uncommon genetic environment of blaNDM-1 was discovered in this study and comparative genomics analysis was performed. The coexistence of blaRSA-1 and blaNDM-1 can result in more extensive antibiotic resistance in the host bacteria, and is a significant threat to public health. This study would not only provide an overall understanding of the genomic characterization of clinically isolated carbapenem-resistant A. caviae, but also provide an in-depth discussion of the multiple acquisition methods of drug resistance genes in Aeromonas. We hope that this study will contribute to the understanding of the drug resistance of Aeromonas, and provide a richer perspective on how drug resistance genes are carried in Aeromonas.In this work, we characterized and deciphered the genomic features and population distribution of 211703, a newly identified extensively drug resistant The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The specimens were obtained with the patient’s consent. The use of human specimens and all related experimental protocols were reviewed and approved by the Ethics Committee of Taizhou Municipal Hospital, Zhejiang, China, in accordance with the medical research regulations of the Ministry of Health, China. Research and all related procedures involving biohazardous materials were approved by the Biosafety Committee of Taizhou Municipal Hospital. This research was conducted in China.Conceptualization, XL, JF, ZY; and FW; methodology, XL, JZ and MX; data analysis, JF, XL, JZ, LY; resources, LY, DH, and PW; writing—original draft, XL and ZY; writing—review and editing, FW and JF. All authors contributed to the article and approved the submitted version.